Although HER2-directed therapies are effective in the subset of breast cancers characterized by amplification of the HER2 gene (HER2+), resistance to these therapies remains an important clinical problem and the mechanisms of this resistance are not well defined. Recent data demonstrate that at least 40% of HER2+ breast cancers have activating mutations in the PI3-kinase gene (PIK3CA) or other alterations in the PI3K pathway. Furthermore, preclinical and clinical investigations, including our own, have implicated these PI3K pathway alterations as potential mediators of resistance to anti-HER2 therapy and have demonstrated that combining PI3K inhibitors with anti-HER2 agents can overcome this resistance. In Project 2, we aim to optimize the application of PI3K-directed therapies, both in novel genetically-engineered mouse (GEM) models and subsequently in a clinical trial. Since a combination of anti-HER2 therapy and PI3K inhibition will likely be ineffective for some patients due to additional mutations bypassing the HER2/PI3K pathway, we will utilize recently developed methods to study mechanisms of resistance that lie outside the PI3K pathway. The aims of this project are to identify and overcome both PI3K-dependent and -independent mechanisms of resistance to targeted therapy of HER2+ breast cancer. Specifically we will: 1) Use GEM models to optimize PI3K-targeted treatment strategies for each subset of HER2+ breast cancer, with an emphasis on comparing pan-PI3K inhibitors with isoform specific agents, 2) Identify novel resistance mechanisms to HER2- and PI3K-targeted therapies in GEM models, and 3) Evaluate the role of PI3K inhibition in conjunction with HER2-targeted therapy in a preoperative clinical trial of patients with HER2+ breast cancer. Tumor tissue from that trial and others will be analyzed with next generation sequencing techniques in order to validate resistance mechanisms identified in Aim 2. Together these studies will strengthen our ability to overcome therapeutic resistance in HER2+ breast cancer and thereby improve outcomes for patients with this disease.